ホ౯఍㆟ሗ࿌᭩human Cell Line Activation Test㸦h-CLAT㸧

JaCVAM ホ౯఍㆟

ᖹᡂ 28 ᖺ㸦2016 ᖺ㸧12 ᭶ 20 ᪥11

JaCVAM ホ౯఍㆟኱㔝Ὀ㞝㸦㐠Ⴀጤဨ఍᥎⸀㸧㸸ᗙ㛗㣤 ሯ ᑦ ᩥ 㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧஬༑ᔒⰋ᫂㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ ⏕ά⾨⏕໬Ꮫ㒊㸧▼ ஭ 㞝 ஧ 㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧஭ୖᬛᙲ㸦᪥ᮏච␿ẘᛶᏛ఍㸧௒஭ᩍᏳ㸦᪥ᮏື≀ᐇ㦂௦᭰ἲᏛ఍㸧ᒾ℩⿱⨾Ꮚ㸦᪥ᮏ〇⸆ᕤᴗ༠఍㸧⠛⏣࿴ಇ㸦⊂❧⾜ᨻἲே ་⸆ရ་⒪ᶵჾ⥲ྜᶵᵓ㸧ᮡᒣ┿⌮Ꮚ㸦᪥ᮏ໬⢝ရᕤᴗ㐃ྜ఍㸧௰஭ಇྖ㸦᪥ᮏ໬Ꮫᕤᴗ༠఍㸧୰ᮧࡿࡾࡇ㸦⊂❧⾜ᨻἲே 〇ရホ౯ᢏ⾡ᇶ┙ᶵᵓ㸧すᕝ⛅ె㸦ᅜ❧་⸆ရ㣗ရ⾨⏕◊✲ᡤ Ᏻ඲ᛶ⏕≀ヨ㦂◊✲ࢭࣥࢱ࣮㸧἟⃝ ⪽㸦᪥ᮏẘᛶᏛ఍㸧᳃⏣ ೺㸦᪥ᮏ⎔ቃኚ␗ཎᏛ఍㸧ᶓ㛵༤㞝㸦᪥ᮏ⓶⭵࢔ࣞࣝࢠ࣮࣭᥋ゐ⓶⭵⅖Ꮫ఍㸧                ௵ᮇ㸸ᖹᡂ 28 ᖺ 4 ᭶ 1 ᪥㹼ᖹᡂ 30 ᖺ 3 ᭶ 31 ᪥22

human Cell Line Activation Test㸦h-CLAT㸧ࡣࠊከࡃࡢ⓶⭵ឤసᛶ≀㉁ࡀᶞ≧⣽⬊ࢆάᛶ໬ࡍࡿࡇ࡜ࢆ฼⏝ࡋࠊᶞ≧⣽⬊ࡢࣔࢹࣝࡢࣄࢺ༢⌫⣔ᇵ㣴⣽⬊ THP-1 ⣽⬊࡟⿕㦂≀㉁ࢆ᭚㟢ࡋࡓ࡜ࡁࡢ⣽⬊⾲㠃ศᏊ
CD86 ࠾ࡼࡧ CD54 ࡢⓎ⌧㔞ࢆᐃࡍࡿࡇ࡜࡟ࡼࡾࠊ⓶⭵ឤసᛶࡢ᭷↓ࢆุᐃࡍࡿヨ㦂ἲ࡛࠶ࡿࠋ

h-CLAT ࡟ࡘ࠸࡚ࡣࠊEuropean Union Reference Laboratory for Alternatives to Animal Testing㸦EURLECVAM㸧࡟ࡼࡿࣂࣜࢹ࣮ࢩࣙࣥ◊✲ࡀᐇ᪋ࡉࢀ 1)ࠊEURL ECVAM Scientific Advisory Committee㸦ESAC㸧࡟ࡼࡿ➨୕⪅ホ౯ࢆཷࡅࡓ 2) ࠋ2016 ᖺ 7 ᭶࡟ࡣࠊOrganisation for Economic Co-operation and Development

OECD ヨ㦂ἲ࢞࢖ࢻࣛ࢖ࣥ Test Guideline: TG㸧442E ࡜ࡋ࡚ᢎㄆࡉࢀࡓ 3)ࠋJaCVAM ホ౯఍㆟ࡣࠊ⓶⭵ឤసᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍࡟ࡼࡾసᡂࡉࢀࡓࠕ⓶⭵ឤసᛶヨ㦂ホ౯ሗ࿌᭩ human Cell Line ActivationTest㸦h-CLAT㸧ࠖ4)ࢆ⏝࠸࡚ࠊᮏヨ㦂ἲࡢጇᙜᛶ࡟ࡘ࠸᳨࡚ウࡋࡓࠋ㸯㸬ヨ㦂ἲࡢᐃ⩏ྡ⛠㸸 human Cell Line Activation Test㸦h-CLAT㸧௦᭰ࡍࡿᑐ㇟ẘᛶヨ㦂㸸 ࣔࣝࣔࢵࢺࢆ⏝࠸ࡿ⓶⭵ឤసᛶヨ㦂㸦OECD TG406㸧࠾ࡼࡧ࣐࢘ࢫࢆ⏝࠸ࡿᒁᡤࣜࣥࣃ⠇ヨ㦂㹙Local Lymph Node Assay㸸LLNA㸦OECD TG429㸧ࠊLLNA㸸DA㸦OECD TG442A㸧ࠊ
LLNA㸸BrdU-ELISA㸦OECD TG442B㸧㹛
ヨ㦂ἲࡢᴫ␎㸸 ᮏヨ㦂ἲ࡛ࡣࠊᶞ≧⣽⬊ࡢࣔࢹࣝ࡜ࡋ࡚ࣄࢺ༢⌫⣔ᇵ㣴⣽⬊࡛࠶ࡿ THP-1 ⣽⬊ࢆ⏝࠸ࠊ⿕㦂≀㉁ࡀ THP-1 ⣽⬊ࢆάᛶ໬ࡍࡿ⬟ຊࢆホ౯ࡍࡿࠋTHP-1 ⣽⬊࡟⿕㦂≀㉁ࢆῧຍࡋ࡚ 24 ᫬㛫ᇵ㣴ࡋࡓᚋࠊfluorescein isothiocyanate㸦FITC㸧࡛⺯ගᶆ㆑ࡋࡓ CD86 ࠾ࡼࡧ CD54 ࡢ≉␗ᢠయࠊ࠾ࡼࡧ
propidium iodide㸦PI㸧ࢆ⏝࠸࡚⣽⬊ࢆᰁⰍࡋࠊࣇ࣮ࣟࢧ࢖ࢺ࣓ࢺ࣮ࣜ࡟ࡼࡾ⣽⬊⾲㠃ศᏊ CD86 ཬࡧCD54 ࡢⓎ⌧㔞ࠊ୪ࡧ࡟⣽⬊⏕Ꮡ⋡ࢆᐃࡍࡿࠋ⿕㦂≀㉁࡛ฎ⌮ࡋࡓ࡜ࡁࡢ CD86 ࠾ࡼࡧ CD54 ࡑࢀࡒࢀࡢᖹᆒ⺯ගᙉᗘࢆࠊ⁐፹ࡢࡳࢆῧຍࡋࡓࢥࣥࢺ࣮ࣟࣝࡢ⺯ගᙉᗘ࡜ẚ㍑ࡋࠊ┦ᑐ⺯ගᙉᗘࢆ⟬ฟࡍࡿࠋ㸰㸬ホ౯࡟⏝࠸ࡓ㈨ᩱ࠾ࡼࡧホ౯ෆᐜࡢ⛉Ꮫⓗጇᙜᛶᮏヨ㦂ἲࡣࠊEURL ECVAM ࡟ࡼࡿࣂࣜࢹ࣮ࢩࣙࣥ◊✲ 1)࡜ࡑࢀ࡟⥆ࡃ ESAC ࡟ࡼࡿ➨୕⪅ホ౯࡟ࡼࡾࠊᐇ㦂ື≀ࢆ⏝࠸ࡓ⓶⭵ឤసᛶヨ㦂ࡢ௦᭰ἲ࡜ࡋ࡚⛉Ꮫⓗ࡟ጇᙜ࡛࠶ࡿ࡜ሗ࿌ࡉࢀ࡚࠾ࡾ 2)ࠊ2016 ᖺ7 ᭶࡟ OECD ヨ㦂ἲ࢞࢖ࢻࣛ࢖ࣥ࡜ࡋ࡚ᢎㄆࡉࢀ࡚࠸ࡿ 3)ࠋJaCVAM ⓶⭵ឤసᛶヨ㦂㈨ᩱ⦅⧩ጤဨ఍࡛
ࡣࠊ⌧ᅾࡲ࡛බ㛤ࡉࢀ࡚࠸ࡿ᝟ሗ 2,3,5-8)ࢆᇶ࡟ᮏヨ㦂ἲࡢ⓶⭵ឤసᛶヨ㦂௦᭰ἲ࡜ࡋ࡚ࡢ⛉Ꮫⓗጇᙜᛶ࡟ࡘ࠸࡚ホ౯ࡋࡓࠋᮏヨ㦂ἲࡣࠊ⓶⭵ឤసᛶⓎ⌧ᶵᗎ࡟࠾ࡅࡿ㔜せ࡞ࢫࢸࢵࣉ࡛࠶ࡿᢠཎᥦ♧⣽⬊ࡢάᛶ໬཯ᛂࢆᣦᶆ࡟ࡋࡓࡶࡢ࡛ࠊάᛶ໬ࡢ㝿࡟ㄆࡵࡽࢀࡿ⣽⬊⾲㠃ศᏊࡢⓎ⌧ஹ㐍ࢆᐃࡍࡿ࡜࠸࠺Ⅼ࡛ཎ⌮ⓗ࡟ࡶጇᙜ࡛࠶ࡿࠋ

33㸱㸬ᮏヨ㦂ἲࡢ᭷⏝ᛶ࡜㐺⏝㝈⏺
ᮏヨ㦂ἲࡣࠊࣄࢺ༢⌫⣔⏤᮶ᇵ㣴⣽⬊ᰴࢆ⏝࠸ࡿ in vitro ヨ㦂ἲ࡛࠶ࡾࠊ3Rs ࡢ⢭⚄࡟ྜ⮴ࡋ࡚࠸ࡿࠋࡲࡓࠊ㸯ヨ㦂࠶ࡓࡾࡢ㈝⏝ࡣࠊ࣐࢘ࢫࢆ⏝࠸ࡿ LLNA ࡢ 1/5 ⛬ᗘ࡛࠶ࡾࠊヨ㦂ᮇ㛫ࡶ LLNA ࡟ẚ࡭࡚▷ᮇ㛫࡛࠶ࡿࡇ࡜࠿ࡽࠊᮏヨ㦂ἲࡣ⤒῭ᛶ࠾ࡼࡧ㎿㏿ᛶࡢ㠃࠿ࡽ᭷⏝࡜࠸࠼ࡿࠋࡋ࠿ࡋ࡞ࡀࡽࠊᮏヨ㦂ἲࡣࠊ⣽⬊ᐦᗘ࡜ᇵ㣴᫬㛫ࢆཝᏲࡍࡿࡇ࡜ࡸබẚ 1.2 ࡢ⃰ᗘタᐃࢆᚲせ࡜ࡍࡿࡓࡵࠊูࡢ⓶⭵ឤసⓎ⌧࢖࣋ࣥࢺࢆᣦᶆ࡜ࡍࡿ Direct Peptide Reactivity Assay㸦OECD TG442C㸧ࡸ ARE-Nrf2 Luciferase Test Method
㸦OECD TG442D㸧࡟ẚ࡭࡚ࠊ᧯సࡀ↹㞧࡛࠶ࡾࠊไ⣙ࡀከ࠸ࠋࡲࡓࠊ㝧ᛶᑐ↷ࠊ㝜ᛶᑐ↷࠾ࡼࡧ፹యᑐ↷ࡢᐃࢆ⥅⥆ⓗ࡟ᐇ᪋ࡋࠊ⫼ᬒࢹ࣮ࢱࢆసᡂࡍࡿࡇ࡜ࡀồࡵࡽࢀ࡚࠸ࡿࠋEURL ECVAM ࡟ࡼࡿࣂࣜࢹ࣮ࢩࣙࣥ◊✲ 1)࡟࠾࠸࡚ࠊ15 ≀㉁ࢆ⏝࠸࡚ᐇ᪋ࡉࢀࡓ᪋タෆ෌⌧ᛶࡣࠊ
73.3㹼86.7%࡛࠶ࡗࡓࡀࠊヨ㦂᫬㛫ࡢཝᐦ࡞⟶⌮࡟ࡼࡾ෌⌧ᛶࡢྥୖࡀᅗࢀࡿ࡜ࡉࢀ࡚࠸ࡿࠋ୍᪉ࠊ24 ≀㉁ࢆ⏝࠸ 4 ᪋タ࡛ᐇ᪋ࡉࢀࡓ᪋タ㛫෌⌧ᛶࡣ 79.2㸣࡛࠶ࡗࡓࠋ୍⮴ࡋ࡞࠿ࡗࡓ≀㉁ࡣ᪋タෆ෌⌧ᛶࡶᝏ࠿ࡗࡓࠋ
EURL ECVAM1)ࠊTakenouchi ࡽ 7)ࠊ࠾ࡼࡧ Nukada ࡽ 8)ࡢ⤖ᯝࢆྜࢃࡏࡓ 142 ≀㉁㸦ឤసᛶ≀㉁㸸㠀ឤసᛶ≀㉁㸻101㸸41ࠊlog Kow ࡀ 3.5 ௨ୖ࡛㝜ᛶ࡜ุᐃࡉࢀࡓ≀㉁࡜ΰྜ≀ࢆ㝖ࡃ㸧࡟ࡘ࠸࡚ࡢᡂ⦼ࡣࠊ
ṇ☜ᗘ 85㸣ࠊឤᗘ 93㸣ࠊ≉␗ᗘ 66㸣࡛࠶ࡾࠊᵝࠎ࡞໬Ꮫ≀㉁࡟㐺⏝ྍ⬟࡛࠶ࡿࡇ࡜ࡀ♧ࡉࢀࡓࠋࡋ࠿
ࡋࠊUnited Nations UN Globally Harmonized System of Classification and Labelling of Chemicals 㸦GHS㸧ศ㢮࡟࠾ࡅࡿ㍍ᗘ㹼୰➼ᗘឤసᛶ≀㉁ࡣഇ㝜ᛶࡢุᐃࡀ⏕ࡌࡸࡍ࠸ഴྥࡀ࠶ࡿࠋࡲࡓࠊヨ㦂⁐፹࡟⁐ゎࡋ࡞࠸≀㉁࠾ࡼࡧヨ㦂⁐፹୰࡛୙Ᏻᐃ࡞≀㉁ࠊ᥹Ⓨᛶ≀㉁ࠊFITC ࡸ PI ࡜ྠࡌἼ㛗ᇦ࡟ᙉ࠸⺯ගࢆ᭷ࡍࡿ≀㉁ࠊࣉࣟࣁࣉࢸࣥ࠾ࡼࡧࣉࣞࣁࣉࢸࣥࡢࡼ࠺࡞άᛶ໬࡟௦ㅰ⣔ࢆᚲせ࡜ࡍࡿ໬Ꮫ≀㉁࡛ࡣṇࡋࡃឤసᛶࡀ᳨ฟࡉࢀ࡞࠸ྍ⬟ᛶࡀ࠶ࡾࠊヨ㦂⤖ᯝࡢゎ㔘࡟ࡣὀពࡀᚲせ࡛࠶ࡿࠋ㸲㸬┠ⓗ࡜ࡍࡿ≀㉁ཪࡣ〇ရࡢẘᛶࢆホ౯ࡍࡿヨ㦂ἲ࡜ࡋ࡚ࡢࠊ♫఍ⓗཷࡅධࢀᛶ࠾ࡼࡧ⾜ᨻୖࡢ฼⏝ࡢྍ⬟ᛶ ♫఍ⓗཷࡅධࢀᛶ㸸ᮏヨ㦂ἲࡣࠊࣇ࣮ࣟࢧ࢖ࢺ࣓ࢺ࣮ࣜ࡟⩦⇍ࡋࡓ᪋タ࡛࠶ࢀࡤᐇ᪋࡛ࡁࡿヨ㦂ἲ࡛࠶ࡾࠊ⺯ගᶆ㆑ࡉࢀࡓ≉␗ᢠయࡶᕷ㈍ࡉࢀ࡚࠸ࡿࠋࡲࡓ THP-1 ⣽⬊ࡢධᡭࡣᐜ࡛᫆࠶ࡾࠊ▷ᮇ㛫࡛Ᏻ౯࡟ᐇ᪋࡛ࡁࡿࠋ⏕ࡁࡓື≀ࢆ⏝࠸࡞࠸࡜࠸࠺Ⅼ࡛ࠊ3Rs ࡢ⢭⚄࡟ྜ⮴ࡋ࡚࠾ࡾࠊ♫఍ⓗཷࡅධࢀᛶࡣ㧗࠸ࠋ ⾜ᨻୖࡢ฼⏝ᛶ㸸ᮏヨ㦂ἲ࡟࠾࠸࡚㝧ᛶࡢ⤖ᯝࡀᚓࡽࢀࡓሙྜࠊឤసᛶ≀㉁࡜ุᐃࡍࡿࡇ࡜ࡣྍ⬟࡛࠶ࡿࡀࠊឤసᛶᙉᗘศ㢮ࡸ UN GSH ࡢࢧࣈ࢝ࢸࢦ࣮ࣜศ㢮ࡣᅔ㞴࡛࠶ࡿࠋᮏヨ㦂ἲ༢⊂࡛ࡢ⓶⭵ឤసᛶࡢุᐃࡣ୙༑ศ࡛࠶ࡾࠊ⿕㦂≀㉁ࡢ≉ᛶࢆ༑ศ࡟⌮ゎࡋࡓୖ࡛ࠊIntegrated Approaches to Testing and Assessment㸦IATA㸧ࢆᵓᡂࡍࡿࡑࡢ௚ࡢ᝟ሗ࡜⤌ࡳྜࢃࡏ࡚㐺ษ࡟ホ౯ࡍࡿࡇ࡜ࡀᚲせ࡛࠶ࡿࠋ࡞࠾ࠊᮏヨ㦂ἲࡢ฼⏝࡟࠶ࡓࡗ࡚ࡣࠊ㐺⏝⠊ᅖࢆ༑ศ࡟㓄៖ࡋࡓୖ࡛౑⏝ࡉࢀࡿ࡭ࡁ࡛࠶ࡿࠋ44ཧ⪃ᩥ⊩1)

EURL ECVAM (2013). human Cell Line Activation Teat (h-CLAT) Validation Study Report. Accessible at:https://eurl-ecvam.jrc.ec.europa.eu/news/news_docs/h-clat-validation-study-report.

2)

EURL ECVAM (2015). Recommendation on the human Cell Line Activation Test (h-CLAT) for skinsensitization testing. Accessible at: https://eurl-ecvam.jrc.ec.europa.eu/news/news_docs/eurl-ecvamrecommendation-on-the-human-cell-line-activation-test-h-clat-for-skin-sensitisation-testing3)OECD (2016). OECD Guideline for the Testing of Chemicals No. 442E: In Vitro Skin Sensitization: humanCell Line Activation Test (h-CLAT). Available at: http://www.oecd-ilibrary.org/environment/test-no-442e-invitro-skin-sensitisation_9789264264359-en;jsessionid=3gdulzg8pwz1.x-oecd-live-024)

JaCVAM⓶⭵ឤసᛶ㈨ᩱ⦅⧩ጤဨ఍㸸⓶⭵ឤసᛶヨ㦂ホ౯ሗ࿌᭩ human Cell Line Activation Test㸦hCLAT㸧㸦2016ᖺ7᭶25᪥㸧㸬5)

EURL ECVAM (2015). Protocol No. 158: human Cell Line Activation Test (h-CLAT). https://ecvamdbalm.jrc.ec.europa.eu/methods-and-protocols/search/h-clat

6)

Ashikaga T, Sakaguchi H, Sono S, Kosaka N, Ishikawa M, Nukada Y, Miyazawa M, Ito Y, Nishiyama N,Itagaki H. (2010). A comparative evaluation of in vitro skin sensitization tests: the human cell-line activation
test (h-CLAT) versus the local lymph node assay (LLNA). Altern. Lab. Anim., 38, 275-284.

7)

Takenouchi O, Miyazawa M, Saito K, Ashikaga T, Sakaguchi H. (2013). Predictive performance of the human
Cell Line Activation Test (h-CLAT) for lipophilic chemicals with high octanol-water partition coefficients. J.
Toxicol. Sci., 38, 599-609.

8)

Nukada Y, Ashikaga T, Miyazawa M, Hirota M, Sakaguchi H, Sasa H, Nishiyama N. (2012). Prediction of skinsensitization potency of chemicals by human Cell Line Activation Test (h-CLAT) and an attempt at classifying
skin sensitization potency. Toxicol. In Vitro, 26, 1150-60.

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